Novel terthiophene-3-carboxylic acid compound and fabricating method thereof, functionalized conductive terthiophene polymer with the compound as a monomer, process for dna hybridizatioon detection using the polymer, and fabricating method of probe dna

ABSTRACT

A novel terthiophene-3-carboxylic acid compound is disclosed that a functionalized conductive terthiophene polymer having carboxylic groups produced in an electrochemical method using the compound as a monomer and a novel process for detecting DNA hybridization through impedance measurement using the polymer. In addition, a method for manufacturing a probe DNA used in the process for detecting DNA hybridization is provided. Since the process can detect DNA hybridization using impedance change, without the use of any indicator, small-sized sensor systems which measure impedance in a particular frequency range can be fabricated. The systems are applicable to portable sensors for identifying DNA sequence hybridization for clinical examination and disease diagnosis. Further, the process has an excellent selectivity of complementary sequences to mismatched sequences.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel process for detecting DNAhybridization through impedance measurement using a novel conductivepolymer in the DNA biosensor and electrochemical biosensor fields andmore particularly to a novel process for detecting DNA hybridization bypreparing a novel 3′-carboxyl-5,2′;5′,2″-terthiophene compound;producing a functionalized conductive terthiophene polymer through anelectrochemical polymerization using the compound as a monomer on aglassy carbon electrode; immobilizing a probe DNA (or oligonucleotide)onto the polymer and measuring the impedance before and after ahybridization with a target DNA (or oligonucleotide).

2. Related Prior Art

Recently, a need has developed for apparatuses capable of continuouslyand selectively detecting biological molecules in the biotechnology andmedical diagnostic fields. In particular, special attention has beenpaid to the development of electrochemical biodetectors or biosensorsbased on DNA hybridization, interactions among drugs and carcinogenicsubstances and DNAs, and special DNA interactions including DNA damagein the electrochemical detection of nucleic acid. Among these sensors, avariety of DNA sensors have been used to detect DNA sequences, toxiccompounds and trace organic compounds. In particular, a hybridizationdetection method for DNA sequences is recognized as one of most reliablemethods in terms of its broad applicability to the genome.

In connection with DNA hybridization detection, some conventionalmethods for detecting DNA hybridization are known. For example, a methodfor optically measuring the intensity of fluorescence after hybridizingan immobilized probe DNA with a fluorescent substance(dye)-labeledtarget DNA (or oligonucleotide), is reported [see, Physicochemical andEngineering Aspects, 2000, 175, 147-152; Analytica Acta, 1997, 350,51-58; Anal. Chem. 1994, 66, 3379-3383].

A method for measuring the redox of an indicator to detect DNAhybridization is also reported [see, Anal. Chem. 1996, 68, 2629-2634;Anal. Chem. 1994, 66, 3830-3833; Anal. Chem. 2000, 72, 1334-1341]. Themethod comprises hybridizing an immobilized probe DNA with a target DNAand reacting the hybridized DNAs with the indicator so that theindicator is intercalated into the double-stranded DNA.

Further, disclosed is a method for measuring frequency change using aquartz crystal microbalance (QCM) when a probe DNA immobilized onto thesurface of electrode is reacted with a target DNA [see, Biointerfaces,1998, 10, 199-204; J. Am. Chem. Soc., 1992, 114, 8299-8300].

Further, a method for measuring a potential shift and current change inthe redox wave of a conductive polymer (polypyrrole probe) when a probeDNA immobilized onto the conductive polymer is reacted with a targetDNA, is disclosed [see, J. Am. Chem. Soc., 1997, 119, 7388-7389;Synthetic Metals, 1999, 100, 89-94].

However, there is a disadvantage in that when a target DNA is labeledwith an indicator, the sample preparation is complicated. In addition,when the indicator is inserted after hybridization, time required forexperimental steps become long and thus industrially disadvantageous.

In the case of the above-mentioned optical method or the method usingQCM, since the measuring instruments used in these methods are huge andexpensive, it is necessary for experienced personnel to manage them, aswell as the fact that they are unsuitable for portable equipment. In theelectrochemical method using a redox indicator, since the degree ofhybridization depends on the sensitivity of the indicator, the method'ssensitivity and selectivity are problematic.

Further, in the case of the method measuring a redox wave of a polymer,the method is inapplicable to portable sensors.

Therefore, there is a need for a portable DNA sensor which can directlydetect DNA hybridization without the use of an indicator therebyshortening the time required in the experiment and which even aninexperienced person can easily identify within a short time whether ornot DNA hybridization has occurred.

Thus, the present inventors have earnestly and widely researched theabove-mentioned problems in the conventional DNA hybridization detectionand have found that when measuring impedance before and afterhybridization using a functionalized conductive terthiophene polymerwith a novel terthiophene-3-carboxylic acid compound as a monomer, DNAhybridization can be directly detected and as a result they accomplishedthe present invention.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a novelcompound 3′-carboxyl-5,2′;5′,2″-terthiophene.

It is another object of the present invention to provide afunctionalized conductive terthiophene polymer produced using the3′-carboxyl-5,2′;5′,2″-terthiophene compound as a monomer.

It is another object of the present invention to provide a novel processfor detecting DNA hybridization by immobilizing a probe DNA (oroligonucleotide) onto the polymer followed by a method for measuringimpedance before and after DNA hybridization.

It is yet another object of the present invention to provide a methodfor manufacturing the probe DNA (or oligonucleotide).

The functionalized conductive terthiophene polymer according to thepresent invention is produced by obtaining 3-cyanoterthiophene from3-bromoterthiophene as a starting material, hydrolyzing the3-cyanoterthiophene to prepare 3′-carboxyl-5,2′;5′,2″-terthiophene andelectrochemically polymerizing the prepared3′-carboxyl-5,2′;5′,2″-terthiophene as a monomer.

The process for detecting DNA hybridization according to the presentinvention comprises the steps of: producing the functionalizedconductive terthiophene polymer through an electrochemicalpolymerization using the terthiophene compound on a glassy carbonelectrode, thereby modifying the carbon electrode with the polymer;immobilizing a probe DNA (or oligonucleotide) onto the conductivepolymer and measuring impedance before and after a hybridization with atarget DNA (or oligonucleotide).

The probe DNA (or oligonucleotide) used in the process for detecting DNAhybridization is manufactured by binding an amine-bound alkyl group tothe 5′-end of a DNA (or oligonucleotide).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the presentinvention will become more apparent from the following detaileddescription when taken in conjunction with the accompanying drawings inwhich:

FIG. 1 is a graph showing a cyclic voltammogram pattern forelectrochemical polymerization of 3′-carboxyl-5,2′;5′,2″-terthiophene asa monomer;

FIG. 2 is a scheme showing an immobilization of a probe oligonucleotideand a hybridization of a target sequence;

FIG. 3 a is a graph showing changes in the impedance(A) andadmittance(B) before and after hybridization on an open circuit voltagein a phosphate buffer (pH 7.4) at room temperature;

FIG. 3 b is a graph showing differences in the impedance before andafter hybridization with a noncomplementary target sequence E and acomplementary target sequence B, respectively; and

FIG. 3 c is a graph showing differences in the impedance before andafter hybridizations with a center base-mismatched sequence and an endbase-mismatched sequence, respectively.

DETAILED DESCRIPTIONS OF THE PREFERRED EMBODIMENT

Hereinafter, the present invention will be explained in more detail.

DNA consists of two strands which form a double helical structure. AnssDNA (single-stranded DNA) binds with another ssDNA having a basesequence complementary to each other to form a double strand. Thisprocess is referred to as hybridization. DNA hybridization detection isa technique determining the differences before and after hybridizationwhen a single-stranded probe DNA immobilized onto the surface of solidsubstrate is hybridized with a target DNA complementary to the probeDNA.

The process for detecting DNA hybridization according to the presentinvention electrochemically detects DNA hybridization by determining thedifference in the impedance before and after hybridization using thefunctionalized conductive terthiophene polymer.

The 3′-carboxyl-5,2′;5′,2″-terthiophene compound according to thepresent invention is represented by the following formula 1:

The functionalized conductive terthiophene polymer according to thepresent invention is produced using the3′-carboxyl-5,2′;5′,2″-terthiophene compound as a monomer. Theterthiophene polymer has conductivity due to its chemical structuralproperty.

Specifically, the functionalized conductive terthiophene polymeraccording to the present invention is produced as follows: first,3-bromoterthiophene as a starting material is reacted with coppercyanide (CuCN) to obtain 3-cyanoterthiophene, represented by thefollowing formula 2:

3-cyanoterthiophene thus obtained has a melting point of 83° C. and amolecular weight of 272.9736 g/mol. Subsequently, 3-cyanoterthiophene ishydrolyzed in the presence of potassium hydroxide-ethoxyethanol toprepare 3′-carboxyl-5,2′;5′,2″-terthiophene of formula 1.3′-carboxyl-5,2′;5′,2″-terthiophene has a melting point of 193° C. and amolecular weight of 292.4002 g/mol. The functionalized conductiveterthiophene polymer according to the present invention is representedby the following formula 3:

The terthiophene polymer is produced in an electrochemical method whichsynthesizes the polymer using 3′-carboxyl-5,2′;5′,2″-terthiophene as amonomer on the surface of an electrode in accordance with cyclicvoltammetry method. The conductive polymer thus prepared has a reddishbrown color.

The process for detecting DNA hybridization using the functionalizedconductive polymer comprises the steps of: dissolving3′-carboxyl-5,2′;5′,2″-terthiophene in a non-aqueous solvent; producingthe conductive terthiophene polymer having carboxylic groups on thesurface of an electrode in an electrochemical method and modifying thepolymer; immobilizing a probe DNA (or oligonucleotide) amino-modified atits 5′-end using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC)onto the polymer; and measuring impedance before and after DNAhybridization.

The electrode used herein is made of glassy carbon and gold. Theterthiophene polymer is electrochemically produced and modified on theelectrode surface in accordance with cyclic voltammetry method, and theprobe DNA (or oligonucleotide) is chemically combined to the carboxylicgroups of the polymer. The probe DNA (or oligonucleotide) used in theprocess for detecting DNA hybridization according to the presentinvention includes those corresponding to a target DNA (oroligonucleotide), and is preferably a 19-mer oligonucleotide. The probeDNA (or oligonucleotide) is a DNA (or oligonucleotide) modified with anamino group at its 5′-end, which is manufactured by binding an alkylgroup having from 3 to 10 carbon atoms, preferably 6 carbon atoms, tothe 5′-end of DNA having base sequences with a certain length, and thencombining an amine to the alkyl group using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) to modify the5′-end into an amino group.

A specific example of the DNA is NH₂—C₆-5′-CTCCTGTGGAGAAGTCTGC-3′.Impedance measurement is performed at a frequency ranging from 10 Hz to100 kHz, and preferably at a frequency of about 1 kHz.

In order to detect DNA hybridization in accordance with the presentinvention, a DNA base sequence from patients suffering from sickle cellanemia is used. First, a single-stranded probe of the DNA base sequenceis immobilized onto the electrode surface. Subsequently, target DNA basesequences (Sequence B: 3′-GAG GACACCTCTTCAGACG-5′, Sequence C:3′-GAGGACTCCTCTTCAGACG-5′, Sequence D: 3′-CTGGACACCTCTTCAGACG-5′,Sequence E: 3′-CCTAGTCTACAGGTCACTA-5′), respectively, complementary tothe single-stranded probe are hybridized in a phosphate buffer. Finally,impedance before and after hybridization is measured. Differencesbetween log values of the measured impedance before and afterhybridization are calculated. The target DNA base sequences include thecompletely complementary sequence B (3′-GAGGACACCTCTTCAGACG-5′), acenter 1-mer mismatched sequence C (3′-GAGGACTCCTCTTCAGACG-5′), an end2-mer mismatched sequence D (3′-CTGGACACCTCTTCAGACG-5′), and acompletely non-complementary sequence E (3′-CCTAGTCTACAGGTCACTA-5′).

The present invention will now be described in more detail withreference to the following examples and drawings.

However, these examples are given by way of illustration and not oflimitation.

EXAMPLE 1 Preparation of 3′-carboxyl-5,2′;5′,2″-terthiophene MonomerHaving carboxyl Groups

Scheme 1

Synthesis of 3-cyanoterthiophene: 10 mmol of 3-bromoterthiophene wasrefluxed in anhydrous dimethylformamide (10 ml) containing 15 mmol ofcopper cyanide for 4 hours. After the dark mixture was allowed to coolto room temperature it was mixed with iron chloride (10 g) inhydrochloric acid solution (20 ml, 2.0M) and maintained at 60˜70° C. for30 minutes. The organic extract was washed with hydrochloric acidsolution (20 ml, 6M), distilled water, saturated sodium bicarbonatesolution and aqueous sodium chloride solution to obtain a dark yellowishsolid. The obtained solid was recrystallized from an organic solvent.The product has a melting point of 83° C. and a molecular weight of272.9736 g/mol as measured using a mass spectrometer. The structure ofthe product was identified through ¹³C NMR, ¹H NMR, and IR spectrometer.

The data are as follows:

¹H NMR (CDCl₃): δ7.06-7.62 (m, 7H); ¹³C NMR δ: 106.0, 115.8, 125.6,127.5, 126.5, 127.7, 128.0, 128.6, 128.8, 133.4, 135.0, 136.7, 145.3

Synthesis of 3′-carboxyl-5,2′;5′,2″-terthiophene: 10 mmol of3-cyanoterthiophene and 17.8 mmol of potassium hydroxide were refluxedin ethoxyethanol-water (5:1) for 5 hours. The reaction mixture wasacidified with an excess of hydrochloric acid solution (12M) and cooleduntil yellowish precipitates were formed. The precipitates werefiltered, washed with distilled water and recrystallized from an organicsolvent to obtain the title product as a solid. The product has amelting point of 193° C., and a molecular weight of 292.4002 g/mol asmeasured using a mass spectrometer. The structure of the product wasidentified through ¹³C NMR, ¹H NMR, and IR spectrometer.

The data are as follows:

¹H NMR (CDCl₃) δ: 7.04-7.59(m, 7H), 7.59 (br s, 1H); ¹³C NMR δ: 125.0,125.9, 126.9, 127.8, 128.4, 128.6, 130.2, 133.6, 135.9, 136.1, 143.8,166.7

EXAMPLE 2 Fabrication of DNA Sensor

Manufacture of a modified electrode using conductive polymer of3′-carboxyl-5,2′;5′,2″-terthiophene having a carboxylic group: After3′-carboxyl-5,2′;5′,2″-terthiophene monomer was dissolved inacetonitrile, the monomer was polymerized on the surface of a glassycarbon and gold electrode in accordance with an electrochemical methodto produce a conductive terthiophene polymer. Subsequently, the polymerwas modified [FIG. 1]. Generally, in order to electrochemically producethe conductive polymer, a voltage, at which a monomer is oxidized, isapplied to a solution containing the monomer, or a cyclic voltammetrymethod by which a polymer is produced on the surface of the electrode,is used. In the present invention, a voltage was repetitively applied ina positive (+) direction, starting at 0.0V and finishing at +1.5V. Atthis time, voltage scan rate was 100 mV/sec. An oxidizing current flowedbetween +1.1V and +1.3V, and a reducing current flowed at +1.15 V. Whenvoltage in a range from 0.0V to +1.5V was cycled, the currentsincreased. Such increase in current demonstrated that the conductivepolymer was produced on the surface of electrode. The number of voltagecycles varied from 1 to 50. FIG. 1 shows cyclic voltammogram pattern at5 voltage cycles. The conductive polymer produced on the surface ofelectrode had a reddish brown color.

Immobilization of probe oligonucleotide: A probe oligonucleotidesequence (NH₂—C₆-5′-CTCCTGTGGAGAAGTCTGC-3′), which is amine-modified atits 5′-end using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC),was immobilized onto an electrode. EDAC used herein is a catalyst tocombine an amino group to a carboxyl group. The conductive polymerelectrochemically produced on the surface of the electrode, inaccordance with the procedure of FIG. 1, has carboxyl groups. Theconductive polymer was combined to a probe oligonucleotide having anamino-modified 5′-end to fabricate a DNA sensor. The electrode coatedwith the conductive polymer, which is produced in accordance with theprocedure of FIG. 1, was immersed in a phosphate buffer (pH 7.4)containing EDAC (10 mg/mL) and the probe oligonucleotide having anamino-modified 5′-end (11 M) and then reacted at room temperature for 10hours to fabricate a DNA sensor. In the DNA sensor, carboxylic acid ofthe conductive polymer was bound with amino group of the probeoligonucleotide. In a similar manner, the binding of a particulardisease-associated probe DNA (or oligonucleotide) with the conductivepolymer will lead to development of disease-associated DNA detectionsensors.

EXAMPLE 3 Impedance Measurement

Impedance measurement was performed before and after hybridizations withtarget DNA sequences in a phosphate buffer. The target DNA base sequenceinclude the completely complementary sequence B(3′-GAGGACACCTCTTCAGACG-5′), a center 1-mer mismatched sequence C(3′-GAGGACTCCTCTTCAGACG-5′), an end 2-mer mismatched sequence D(3′-CTGGACACCTCTTCAGACG-5′), and a completely non-complementary sequenceE (3′-CCTAGTCTACAGGTCACTA-5′) [FIGS. 3 a to 3 c]. FIG. 3 a showsimpedances of the probe oligonucleotide (a single-stranded DNA sequenceimmobilized onto the conductive polymer on the electrode surface) in thefrequency range from 100 kHz to 10 Hz in a phosphate buffer (pH 7.4)containing 0.75M NaCl after the probe oligonucleotide was immobilizedonto the electrode surface and impedances were measured under the sameconditions as above after the electrode was reacted with target DNAsequences (single-stranded DNA sequences to be reacted with the probe)for 30 minutes. Following reacting with the target DNA sequences,impedance values sharply decreased (see, (A) in FIG. 3 a). It can alsobe seen that there were significant differences in admittancemeasurement before and after hybridization (see, (B) in FIG. 3 a). Asshown in FIG. 3 a, resistance (y-axis) dropped after hybridization. Thismeans that conductivity increased after hybridization.

After hybridizing with the non-complementary target sequence E, a graphshowing differences between log values of the measured impedances beforeand after hybridization was plotted (see, FIG. 3 b). As shown in FIG. 3b, the differences were close to zero. This suggests that since nohybridization occurred there was no change in impedances afterhybridization. However, when the electrode was again hybridized with thecompletely complementary target DNA sequence B, there were significantdifferences (FIG. 3 b). This suggests that hybridization occurred andthus there was significant change in impedance after hybridization.Accordingly, it was possible to distinguish the completely complementarytarget DNA sequence B from the completely non-complementary sequence E.

After hybridizing the center 1-mer mismatched sequence C (see, uppergraph in FIG. 3 c) and the end 2-mer mismatched sequence D (see, lowergraph in FIG. 3 c), a graph showing differences between log values ofthe measured impedances before and after hybridization was plotted. Theresults were obtained through 3 repetitive measurements for eachsequence. After hybridization, impedance change was slight, butcomparable to the completely complementary sequence (Sequence B).Accordingly, it was possible to distinguish the completely complementarysequence from the mismatched sequences through impedance measurement.

As shown in FIG. 3, impedance change was greatest at 1 kHz, followinghybridization with the target DNA sequences and the process fordetecting DNA hybridization according to the present invention had anexcellent selectivity to mismatched base sequences, compared toconventional detecting methods. Accordingly, the process for detectingDNA hybridization according to the present invention can simply identifywhether or not hybridization is done through impedance measurement at acertain frequency, for example 1 kHz.

Experimental Results:

Selectivity depends on the ability to distinguish the completelycomplementary sequence from mismatched sequences. In the presentinvention, the degree of hybridization was evaluated by comparing logvalues of the measured impedances before and after hybridization at 1kHz. From the evaluated degree of hybridization, selectivity to aparticular sequence can be seen.

The results are shown in Table 1 below. TABLE 1 |Z|/10 n Degree ofOligonucleotide Sequences^(b) Ohm Hybridization (%)^(c) Complementary(Sequence B) 0.7 ± 0.05  100% 3′-GAG-GAC-ACC-TCT-TCA-GAC-G-5 1-basemismatched sequence (Sequence C) 0.1 ± 0.05 14.3%3′-GAG-GAC-TCC-TCT-TCA-GAC-G-5′ End 2-base mismatched sequence (SequenceD) 0.1 ± 0.05 14.3% 3′-CTG-GAC-ACC-TCT-TCA-GAC-G-5′ Non-complementary(Sequence E) 0.0 ± 0.05   0% 3′-CCT-AGT-CTA-CAG-GTC-ACT-A-5′^(a)probe oligonucleotide of NH₂-C₆-5′-CTC-CTG-TGG-AGA-AGT-CTG-C-3 wasimmobilized onto the polyterthiophene-modified glass carbon electrode.^(b)Concentration: 110 nmol in 10 ml^(c)denotes the amount of oligonucleotides combined to the probeimmobilized onto the modified electrode.

Comparison with Known Data:

In 1992, Okahata et al. performed hybridization detection on various10-mer nucleotides using QCM. In the experiment, the end 2-mermismatched sequence showed a hybridization of 92%, and the centermonomer mismatched sequence showed a hybridization of about 30% (see,Table 2). On the other hand, as shown in Table 1, the process fordetecting hybridization according to the present invention showed ahybridization of 14.3%. Accordingly, it can be seen that hybridizationdetection using impedance can selectively distinguish hybridizationswith target DNA sequences. The degree of hybridization between aprobe-immobilized QCM and various 10-mer nucleotides at 25° C. is shownin Table 2 below. TABLE 2 Degree of hybridization Nucleotides^(b) Δm/ng(%) 3′dCCCTTAAGCA5′ 380 ± 10 100 3′dCCCTTAAGGG5′ 350 ± 10 923′dTGCTTAAGCA5′ 350 ± 10 92 3′dCCCTAAAGCA5′ 125 ± 10 31 3′dCCCTGAAGCA5′100 ± 10 26 3′dCTGCTACGGG5′ 0 0 3′dAGCCGTACCC5′ 0 0^(a)HS-^(5′)PdGGGAATTCGT ^(3′) was immobilized onto QCM in a relativelylarge amount (847 ng, 280 pmol, ca. 65% of electrode coating) in orderto obtain high sensitivity to oligonucleotides having a relatively lowmolecular weight, compared to M13 phage DNA,^(b)Concentration: 400 ng in 10 ml (130 pmol). The underlines denotesequences complementary to the probe.^(c)denotes the amount of oligonucleotides combined to the probeimmobilized on QCM.

As can be seen from the foregoing, according to the present invention, aprocess capable of detecting DNA hybridization using impedance change,without the use of any indicator, is provided. Therefore, small-sizedsensor systems which measure impedance in a particular frequency rangecan be fabricated. The systems are applicable to portable sensors foridentifying DNA sequence hybridization for clinical examination anddisease diagnosis.

Further, the process for detecting DNA hybridization according to thepresent invention has an excellent selectivity of complementarysequences to mismatched sequences.

While this invention has been described in connection with what ispresently considered to be the most practical and preferred embodiment,it is to be understood that the invention is not limited to thedisclosed embodiment and the drawings, but, on the contrary, it isintended to cover various modifications and variations within the spiritand scope of the appended claims.

1. 3′-carboxyl-5,2′;5′,2″-terthiophene represented by the followingformula 1:


2. A method for preparing 3′-carboxyl-5,2′;5′,2″-terthiophene accordingto claim 1 comprising the steps of treating 3-bromoterthiophene withcopper cyanide (CuCN) to obtain 3-cyanoterthiophene, and hydrolyzing theobtained 3-cyanoterthiophene in KOH-ethoxyethanol.
 3. A functionalizedconductive terthiophene polymer of the following formula 3 produced inan electrochemical method which synthesizes the polymer using3′-carboxyl-5,2′;5′,2″-terthiophene as a monomer on the surface ofelectrode in accordance with cyclic voltammetry method:


4. A process for detecting DNA hybridization comprising the steps of:dissolving 3′-carboxyl-5,2′;5′,2″-terthiophene in a non-aqueous solvent;producing the conductive terthiophene polymer having carboxylic groupson the surface of electrode in an electrochemical method and modifyingthe polymer; immobilizing a probe DNA amino-modified at its 5′-end using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) onto the polymer;and measuring impedance before and after DNA hybridization.
 5. Theprocess for detecting DNA hybridization according to claim 4, whereinthe impedance measurement is performed at a frequency of 1 kHz.
 6. Amethod for manufacturing a probe DNA comprising the steps of: binding analkyl group having from 3 to 10 carbon atoms to the 5′-end of DNA havingbase sequences with a certain length; and combining an amino group tothe alkyl group using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDAC) to modify the 5′-end into an amino group.
 7. The method formanufacturing a probe DNA according to claim 6, wherein the DNA is a19-mer oligonucleotide, and the alkyl group has 6 carbon atoms.